Hepatitis C virus (HCV) is the major etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It is estimated that over 100 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, so called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death.
The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been thoroughly elucidated. It is not known how HCV interacts with and evades the host immune system. In addition, the roles of cellular and humoral immune responses in protection against HCV infection and disease have yet to be established. Various clinical studies have been conducted with the goal of identifying pharmaceutical compounds capable of effectively treating HCV infection in patients afflicted with chronic hepatitis C. These studies have involved the use of interferon-alpha, alone and in combination with other antiviral agents such as ribavirin. Such studies have shown that a substantial number of the participants do not respond to these therapies, and of those that do respond favorably, a large proportion were found to relapse after termination of treatment. To date there are no broadly effective antiviral compounds for treatment of HCV infection. HCV is an enveloped positive strand RNA virus in the Flaviviridae family.
The single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce structural and non-structural (NS) proteins. The structural proteins (C, E1, E2 and E2-p7) comprise polypeptides that constitute the virus particle. The non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) encode for enzymes or accessory factors that catalyze and regulate the replication of the HCV RNA genome. Processing of the structural proteins is catalyzed by host cell proteases. The generation of the mature non-structural proteins is catalyzed by two virally encoded proteases. The first is the NS2–3 zinc-dependent metalloprotease which auto-catalyses the release of the NS3 protein from the polyprotein. The released NS3 contains a serine protease domain at the N-terminal and catalyzes the remaining cleavages from the polyprotein. The released NS4A protein has at least two roles. First, forming a stable complex with NS3 protein and assisting in the membrane localization of the NS3/NS4A complex (Kim et al., 1999) and second, acting as a cofactor for NS3 protease activity. This membrane-associated complex, in turn catalyzes the cleavage of the remaining sites on the polyprotein, thus effecting the release of NS4B, NS5A and NS5B. The C-terminal segment of the NS3 protein also harbors nucleoside triphosphatase and RNA helicase activity. NS5B is an RNA-dependent RNA polymerase that is involved in the replication of HCV.
The N-terminal 180 amino acids of the NS3 protein is a trypsin-like serine protease that mediates as a first step the auto-cleavage of NS3/4A. The membrane associated NS3/4A complex further cleaves NS4A/4B, NS4B/5A, and NS5A/NS5B junctions to release the viral enzymes considered essential for viral replication. The complexing of NS3/NS4A is an important step in the sequential downstream processing of the polyprotein. The NS3 protein is the most thoroughly characterized HCV protein. Kinetic parameters for cleavage of all the processing sites have been described. Both the N-terminal protease and C-terminal helicase domains have been independently crystallized and high resolution three-dimensional models exist for these structures.
NS3 protease activity is an attractive target for drug discovery. Enzymatic studies have shown that peptides based on the N-terminal product of the NS5A/5B cleavage site are competitive inhibitors of the enzyme. These peptides have served as a useful starting point in medicinal chemistry efforts to rationally design NS3 protease inhibitors as clinically effective anti-HCV compounds.
Due to the high incidence of HCV and the consequences of HCV infections, finding therapeutic compounds against HCV has become important. To that end, efforts to discover compounds against HCV have necessitated developing assay systems for screening and selecting anti-HCV compounds.
To date, a convenient cell culture replication system for the HCV virus is not available. This deficiency has restricted most of the HCV inhibitor screening to in-vitro enzymatic assays and indirect surrogate cell-based assay (Lohmann et al. (1999)). This has severely limited the evaluation of potential anti-HCV compounds in cell culture.
Hirowatari et al. (1995) describes the co-transfection of a first plasmid containing the NS2/NS3 domain fused to the NS5A/NS5B cleavage site (substrate for NS3) and the transactivator Tax 1, and a second plasmid containing a reporter gene whose expression is dependent on Tax 1 transactivation. The NS5A/5B site is cleaved by the expressed NS2 or NS3 protease activity thereby releasing Tax 1 to transactivate the reporter gene on the second plasmid. Thus, the amount of the expressed reporter gene is a measure of NS2 or NS3 proteolytic activity. One of the drawbacks is that this system does not distinguish between the activities of the NS2 metalloprotease and NS3 protease activity. In addition, this system does not allow the measure of protease activity of the NS3 protein when complexed with the NS4A domain, a complex that enhances the specificity of the protease. This assay also does not allow one to measure the protease activity in a system that is closest to the natural context of polyprotein processing. Finally, a further drawback resides in the fact that this system is semi-quantitative, therefore not suitable for quantitative high-throughput screening.
Overton, H. et al. (1995), teach a baculovirus-expressed HCV NS3 activity in insect cells. A series of baculovirus constructs designed to express NS3 protease activity and different substrates are described. Constructs encoding partial NS2/NS3 and NS3/NS4A/NS4B are transfected into an insect cell line. Additional viral constructs encoding NS3 to NS5A and NS5A/5B are transfected alone or together with one of the above constructs. The expressed and cleaved products are visualized immunologically by Western analysis. Some of the deficiencies in this system relate to the use of an insect instead of a mammalian cell line, as well as the requirement for viral infection, an event that may further disrupt the normal functions of the cell. This system also employs the T7 polymerase expression system, a system that is not necessary for the system of the present invention. In addition, the method for detection of the cleavage products is lengthy, difficult to quantify with precision and not amenable to high throughput scale.
Song et al., (1996) described a protease assay system utilizing a lexA-GAL4 fusion protein in yeast. The authors describe inserting the NS3 protease and a cleavage site between the lexA-DNA binding domain and the transcriptional activating domain of GAL4. Cleavage of that site by NS3 protease renders GAL4 transcriptionally inactive leading to the inability of the transformed yeast to synthesize β-galactosidase. This system lacks the NS4A and does not reproduce processing in the context of the HCV polyprotein.
Cho, Y. G. et al., (1997), teach an assay using the sindbis viral replication system. This hybrid virus construct encodes the HCV NS3/NS4A protease region linked to SIN core proteins by the NS4A/NS4B cleavage sequence. Assembly of viral particles in a mammalian cell line is dependent on the processing of the NS4A/4B cleavage site. One major drawback of this system resides in that the HCV protease activity produces chimeric virus that induce cytopathic morphological changes in cells. Measurement of the pH change of the media constitutes a semi-quantitative way of measuring these changes at best. A further drawback in this system is that the sindbis core protein contains a natural serine protease cleavage site similar to the NS3 site thus making this system of limited use for screening potential protease inhibitors. Cho, Y. G. et al., (1998), teach an expression vector encoding the NS3/NS4A region and the NS4A/4B cleavage site fused to the SEAP (secreted alkaline phosphatase) gene, transfected into a mammalian cell line. Cleavage of the NS4A/4B site by NS3 protease releases SEAP protein into the media. The amount of SEAP protein in the media is a measure of NS3 protease activity. One drawback of this system lies in the fact that the reporter molecule is directly fused to the substrate protein and may therefore affect the natural conformation of viral complex proteins (NS3/NS4A). A further major drawback resides in the fact that the amount of reporter protein secreted is in direct proportion to the amount of protein expressed and cleaved in the system (1 substrate molecule cleaved=1 reporter molecule secreted). Since the HCV polyprotein is a system that is expressed at very low levels (even in its natural context), the signal observed is too low to be carried out on a large screening scale.
WO 98/00548, describes hybrid viruses comprising a picornavirus, preferably poliovirus, HCV NS3 protease domain and a single NS3 protease target site. These chimeric viruses are engineered such that the proteolytic processing activity of HCV NS3 is essential for viral viability and proliferation. Various hybrids, each having a different NS3 cleavage site (NS5A/NS5B, NS4A/NS4B or NS4B/NS5A) are taught. Viability, is measured by the viral titer using the plaque assay on HeLa monolayer cells. Once again this system does not provide quantifiable means for screening large numbers of potential inhibitors in a high throughput fashion and does not provide for screening of NS3 protease activity in the natural context of the polyprotein segment.
U.S. Pat. No. 5,861,267 by Vertex discloses a method for assaying HCV NS3 protease that utilizes expression of the NS3/NS4A region and the NS4A/NS4B cleavage site fused to the secreted IL-1β reporter (interleukin-1β). Cleavage at the NS4A/4B site by the NS3 protease releases IL-1β into the medium which permits a direct measure of NS3 protease activity. This system examines inhibition of NS3 cleavage at a cleavage site adjacent to the NS3/NS4A complex, a situation that does not represent or replicate the authentic conditions of multiple sites of polyprotein processing present in infected cells. In addition, this system provides a reporter system where the signal measured is in direct proportion to the amount of protein expressed in the system and the amount of protein cleaved, once again giving rise to a signal too low to be carried out on a large screening scale.
WO 00/08469 by Agouron discloses a further system comprising a protease-reporter construct consisting of the NS2 metalloprotease, the NS3 protease, the NS4A co-factor and different variations of truncated NS4B and 5A, preceding the NS5A/5B cleavage site. This system however, does not disclose the importance of having a full polyprotein for optimizing the NS3 protease activity/specificity. In addition, this system requires infection by a vaccinia virus vector, a factor that mitigates the host cell integrity and may affect the mechanism of cis or trans protease cleavage during polyprotein processing events in the assay. A further drawback of using the vaccinia expression lies in the fact that the cells in the assay become necrotic after about 24 hours, thereby limiting the use of this assay for longer term kinetic assays.
WO 00/12727 by Vertex discloses a system having a fusion protein comprising a ligand binding domain, a DNA binding domain that can bind to a ligand-response element causing the VP16 activation domain to regulate expression of a reporter gene. A NS5A/5B cleavage site is inserted within that fusion protein and modulates the reporter gene expression upon cleavage by the NS3/4A protease which is expressed from a separate construct. Once again, this system does not disclose the importance of having a full polyprotein for optimizing the NS3 protease activity/specificity.
It has therefore become important to develop an assay for screening large numbers of anti-HCV compounds, with the capacity to scale up to a high throughput system. The present invention therefore provides an assay that is easy to perform, reliable, sensitive and reproducible on large scale.
It is therefore the purpose of this invention to provide a cell-based system and assay having improved sensitivity for measuring inhibition of the HCV NS3 protease activity, this assay being designed to concurrently test the protease activity in a construct that reproduces as much as possible the NS3 polyprotein processing events occurring in infected cells in the course of HCV disease.
The present application refers to a number of documents, the content of which is herein incorporated by reference.